Breast cancer is the second leading cause of cancer deaths in women, despite a number of treatment options. Most breast cancers are estrogen receptor-positive and commonly treated with anti-estrogens, while antibodies and kinase inhibitors are used to treat cancers that overexpress the HER2 receptor tyrosine kinase. Unfortunately, tumors are often intrinsically resistant or develop resistance to these drugs due to overexpression of specific genes. One of these genes is the scaffolding protein BCAR1 (breast cancer anti- estrogen resistance 1 or p130Cas), which is a well-known key component of the signaling pathways that regulate cell proliferation, survival and migration/invasion downstream of integrin adhesion receptors. BCAR1 is also a critical node in HER2 oncogenic signaling pathways and mediator of resistance to adriamycin, a drug frequently used to treat breast cancers that do not respond to anti-estrogens or HER2-targeted therapies. Thus, BCAR1 is a central player in the signaling networks that control breast cancer malignancy. Another important but poorly characterized factor in anti-estrogen resistance is the SH2 domain-containing protein BCAR3, a member of a family of three proteins that also includes SHEP1, which we identified in a screen for Eph receptor tyrosine kinase-binding molecules. Members of the BCAR1 and BCAR3 families function in a concerted manner through direct association of their C-terminal regions, but the structure of their complexes has eluded investigators. To gain insight into these crucial yet enigmatic assemblies, we have solved the first crystal structure of a BCAR1-BCAR3 family complex, that of BCAR1 and SHEP1, revealing a novel type of protein interaction. The SHEP1 C-terminal region overall resembles a Cdc25-type guanine nucleotide exchange factor domain. However, crucial regions are altered by BCAR1 binding, resulting in a "closed" conformation of SHEP1 that cannot bind Ras GTPases. We now seek to unravel the mechanistic basis of breast cancer malignancy and resistance to chemotherapy mediated by BCAR1 association with BCAR3, a protein related to SHEP1 but with distinct differences in key features. Thus, we propose a multidisciplinary approach that integrates a spectrum of effective tools ranging from high resolution structural and biochemical analysis to functional studies in engineered cancer cells in culture and mouse breast cancer xenograft models. Our goals will be accomplished by (1) unraveling the molecular details of the BCAR1-BCAR3 signaling association to precisely define the regulatory modifications occurring upon complex formation;and (2) elucidating the role of the BCAR-BCAR3 association in breast cancer growth, invasiveness and resistance to chemotherapy. Our studies will lead to a new understanding of the BCAR1-BCAR3 signaling node as well as complexes of related proteins, and shed light on how breast cancer cells acquire the malignant characteristics that enable them to metastasize and cope with chemotherapeutic insults. Thus, this work will ultimately provide a new basis for developing treatment options to overcome cancer drug resistance. PUBLIC HEALTH RELEVANCE: Our long-term goal is to understand in detail how members of the BCAR1 and BCAR3 protein families promote breast cancer malignancy by tightly binding to each other. Given its central role in breast cancer, our work on the BCAR1-BCAR3 signaling complex will be of particular importance for understanding mechanisms that enable cancer cells to metastasize and resist chemotherapeutic treatments, which are the two main causes of the high mortality associated with breast cancer. Besides providing new valuable knowledge, the information obtained will also be instrumental for designing treatments that target BCAR1-BCAR3 family complexes to sensitize malignant breast cancer cells to existing chemotherapeutic agents.